1. Field of the Invention
The present invention relates generally to a method for improving the specificity of immunoassays, and more particularly, relates to a method for adding superoxide dismutase to a specimen diluent to increase the specificity of immunoassays which utilize recombinant antigens expressed as a fusion protein with superoxide dismutase.
2. Description of Related Art
Historically, immunoassays have been known to be prone to false positive reactions. Viral lysates or recombinant protein preparations, which comprise the antigen solutions which are utilized on the solid support, can also contain other proteins. These other proteins can bind to the solid support, introducing the possibility of reactivity with antibodies in the patient's sample. Alternatively, a component in the patient's sample can bind to the solid support and interfere with the assay.
For example, McFarlane et al., Lancet (1990) 335:754-57, reported a high prevalence of antibody to hepatitis C virus (HCV) in patients with autoimmune chronic active hepatitis (AI-CAH). They suggested that the serum of AI-CAH patients may contain a component that gives false-positive results in the assay. McFarlane et al. surmised that the assay might have been non-specifically detecting IgG since they saw a correlation between IgG levels and OD values, or that factors contained in the patient sera were responsible for the results, such as an antibody against some other pathogen which cross-reacts with the antigen used in the assay, or a component which adheres to the solid phase and binds IgG.
In addition, some false positive results are due to cross-reactivity between the patient sera and the fusion protein used to express the recombinant antigen utilized in the assay. For example, components in patient sera can react with E. coli proteins used in recombinant HIV assays. This reactivity can be expected because E. coli organisms, which constitute the majority of the intestinal flora of man, are foreign to the human body, and the human immune system mounts an immunogenic response against the existant antigen.
On the other hand, it normally is unexpected that humans would possess antibodies against superoxide dismutase (SOD). Superoxide dismutases are enzymes which exist in some form in almost every living organism. In humans, superoxide dismutase catalyzes the conversion of superoxide (O.sub.2 -) to oxygen and H.sub.2 O.sub.2 (Fridovich, Adv. Enzymol. (1974) 41:35). Phagocytes use superoxide and other microbicidal oxidants to destroy ingested microorganisms. These oxidants are produced during phagocytosis and are characterized by unusually high reactivity and the presence of an unpaired electron. If left unchecked, the oxidants may damage host cells and tissues; however, phagocytes possess several means of defending themselves against the endogenously produced oxidants. One of these means is the enzyme superoxide dismutase, which scavenges superoxide before it can harm host cells and tissues.
There is a substantial need, therefore, for an improved method for detecting antibodies by increasing assay specificity.